Methods of screening for compounds that inhibit the biosynthesis of gpi in malaria parasites

ABSTRACT

The present inventors succeeded in isolating GWT1 (PfGWT1), which is one of the enzymes involved in GPI biosynthesis in the malaria parasite  P. falciparum . In addition, the inventors revealed that degenerate mutant DNAs, with a lower AT content than the DNA encoding the PfGWT1 protein, can complement the phenotype of GWT 1 -deficient yeast. Based on the findings, the present invention provides the GWT1 protein of malaria parasites and the use of the protein in methods of screening for antimalarial drugs. The present invention also provides degenerate mutant DNAs encoding proteins involved in GPI biosynthesis, and which have a lower AT content than the original DNAs. The present invention also provides methods of screening for antimalarial drugs which use the degenerate mutant DNAs.

TECHNICAL FIELD

The present invention relates to methods of screening for compounds thatinhibit the biosynthesis of GPI in malaria parasites.

BACKGROUND ART

Malaria is the most common infectious human disease caused by parasiticprotozoans. The disease is caused by infection with malaria parasitesand is mediated by the mosquito, Anopheles gambiae. Every year there areestimated 500 million cases of malaria infection, including more thantwo million fatal cases (Gardner, et al., Nature 419:498-511, 2003). Atpresent 40% of the world's population lives in malaria-epidemic areas,where it is said that one in every three infants dies from malaria.

Glycosylphosphatidylinositol (GPI) is known to play a key role in thegrowth and infectivity of parasites, including malaria parasites. Thereare many GPI-anchored proteins on the cell surface of these parasites.GPI-anchored proteins include proteins such as MSP-1 that function whenthe parasites invade red blood cells. GPI proteins act as parasiticantigens and initiate an immune response in the host. Thus, they havelong been the subject of research aimed at vaccine development.

GPI not only functions as an anchor to tether proteins to the cellmembrane, but is also an abundant glycolipid component of malariaparasite cell membranes. Recent studies have revealed that GPI is toxicand causes lethal symptoms. GPI induces the expression of inflammatorycytokines such as TNF-α, and of adhesion molecules. As a result,infected red blood cells adhere to capillaries, obstructing vessels(sequestration), brain blood vessels in particular. This induces furtherinflammatory reactions that are believed to lead to encephalopathy. Veryrecently, Schofield et al. reported that an anti-GPI antibody reduceslethality in an in vivo infection model system using the murine malariaparasite Plasmodium berghei, and that in vitro, the antibody inhibitslate inflammatory reactions caused by Plasmodium falciparum (SchofieldL, et al., Nature 418:785-789, 2002). These findings suggest that GPI isa major factor in the lethality of malarial infections.

It has also been reported that the acylation of inositol is essentialfor binding mannose to GPI (Gerold, P. et al., Biochem. J. 344:731-738,1999), and that the inhibition of inositol acylation, caused by excessglucosamine, inhibits the maturation of the malaria parasite P.falciparum (Naik, R. S. et al., J. Biol. Chem. 278:2036-2042, 2003).Thus, compounds that can selectively inhibit GPI biosynthesis,particularly the acylation of inositol, may be highly usefulantimalarial drugs.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide antimalarial drugsthat inhibit the biosynthesis of GPI. More specifically, the presentinvention provides the malaria parasite DNA that encodes the GWT1protein, which is a protein involved in the biosynthesis of GPI (GPIsynthase). The present invention also provides a method of using thisDNA in methods of screening for antimalarial drugs. The presentinvention also provides degenerate mutant DNAs of the DNA that encodesthe malaria parasite GPI biosynthesis protein. These degenerate mutantDNAs have a lower AT content than the original DNA. The presentinvention also provides a method of using the degenerate mutant DNAs inmethods of screening for antimalarial drugs.

The GWT1 gene was originally found to encode a fungal GPI-anchoredprotein synthase (WO 02/04626), and is conserved in organisms rangingfrom yeasts to humans. The present inventors confirmed that GWT1homologues (PfGWT1 for P. falciparum GWT1; PyGWT1 for P. yoelii yoeliiGWT1) are included in the entire genomic sequences of Plasmodiumfalciparum (P. falciparum) and Plasmodium yoelii yoelii (P. yoeliiyoelii) (Gardner, et al., Nature 419:498-511, 2003; Carlton et al.,Nature 419:512-519, 2003). The present inventors also found that theGWT1 gene product acts as a GlcN-PI acyltransferase in the GPIbiosynthesis pathway. The PfGWT1 gene product is expected to havesimilar activity, and thus compounds that inhibit this activity can bepromising antimalarial drugs.

In WO 02/04626, the present inventors disclosed a group of compoundsthat inhibit the activity of the fungal GWT1 gene product. Compoundsinhibiting the activity of the PfGWT1 gene product were expected to beantimalarial drugs.

In the present invention, the present inventors succeeded in isolating aregion thought to be almost the full length of the PfGWT1. Using theGWT1 gene products of malaria parasites such as P. falciparum,antimalarial drugs can be screened through binding assays,glucosaminyl(acyl)phosphatidylinositol (PI-GlcN) acyltransferase assays,or using GPI-anchored protein detection systems. Compounds obtained fromsuch screenings can be promising antimalarial drugs. Furthermore, thepresent inventors revealed that degenerate mutant DNAs (degeneratemutants of the DNA that encodes the malaria parasite GPI biosynthesisprotein) having a lower AT content than the original DNA, complement thephenotype of the GWT1 gene-deficient fungus. Thus, it is possible toscreen for compounds that inhibit the activity of proteins involved inGPI biosynthesis in malarial parasites by using, as an index, thephenotype of a GPI synthase gene-deficient fungus, into which adegenerate mutant DNA with a lower AT content (than the DNA encoding theGPI biosynthesis protein in malaria parasites) has been introduced.

Specifically, the present invention provides the following [1] to [25]:

[1] a DNA according to any one of (a) to (d), which encodes a protein ofa malaria parasite having a GlcN-PI acyltransferase activity:

(a) a DNA encoding a protein comprising the amino acid sequence of SEQID NO: 2 or 4,

(b) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3,

(c) a DNA hybridizing to a DNA comprising the nucleotide sequence of SEQID NO: 1 or 3 under stringent conditions, and

(d) a DNA encoding a protein which comprises the amino acid sequence ofSEQ ID NO: 2 or 4, in which one or more amino acids have been added,deleted, substituted, and/or inserted;

[2] a protein encoded by the DNA according to [1];

[3] a vector into which the DNA according to [1] is inserted;

[4] a transformant which retains, in an expressible state, the DNAaccording to [1] or the vector according to [3 ]

[5] an antimalarial drug which comprises as an active ingredient acompound that inhibits the activity of the protein according to [2];

[6] the antimalarial drug according to [5], wherein the compound thatinhibits the activity of the protein according to [2] is at least oneselected from the group consisting of the following compounds (1) to(5):

[7] a method of screening for a compound having antimalarial activity,which comprises the steps of:

(1) contacting the protein according to [2] with a test sample and alabeled compound that has the activity of binding to the protein,

(2) detecting the labeled compound that binds to the protein, and,

(3) selecting a test sample that decreases the amount of labeledcompound that binds to the protein;

[8] the method according to [7], wherein the labeled compound that hasthe activity of binding to the protein is produced by labeling at leastone compound selected from the group consisting of the compounds (1) to(5) according to [6];

[9] a method of screening for a compound having antimalarial activity,which comprises the steps of:

(1) contacting a test sample with the protein according to [2],

(2) detecting GlcN-(acyl)PI, and,

(3) selecting a test compound that decreases the level of GlcN- (acyl)PI;

[10] a method of screening for a compound having antimalarial activity,which comprises the steps of:

(1) contacting a test sample with a cell overexpressing the proteinaccording to [2],

(2) determining the amount of GPI-anchored protein transported to thecell wall, and,

(3) selecting a test sample that decreases the amount of theGPI-anchored protein transported to the cell wall, as determined in step(2);

[11] a method for treating malaria, which comprises administering acompound that inhibits the activity of the protein according to [2];

[12] the method according to [11], wherein the compound that inhibitsthe activity of the protein according to [2] is the compound accordingto [5];

[13] a DNA encoding a protein that has the activity of complementing thephenotype of a GPI synthase gene-deficient yeast, which is a degeneratemutant of a DNA encoding a protein involved in GPI biosynthesis inmalaria parasites, and that has a lower AT content than the originalDNA;

[14] a DNA encoding a protein that has the activity of complementing thephenotype of a GPI synthase gene-deficient yeast, which is a degeneratemutant of a DNA encoding a protein involved in GPI biosynthesis inmalaria parasites, and that has an AT content which is reduced by 70%;

[15] the DNA according t6 [13] or [14], which is selected from the groupconsisting of:

(a) a DNA encoding a protein that comprises any one of the amino acidsequences of SEQ ID NOs: 2 and 4, and odd sequence identificationnumbers in SEQ ID NOs: 6-47,

(b) a DNA comprising any one of the nucleotide sequences of SEQ ID NOs:1 and 3, and even sequence identification numbers in—SEQ ID NOs: 6-47,

(c) a DNA hybridizing under stringent conditions to a DNA that comprisesany one of the nucleotide sequences of SEQ ID NOs: 1 and 3, and evensequence identification numbers in SEQ ID NOs: 6-47, and,

(d) a DNA encoding a protein which comprises any one of the amino acidsequences of SEQ ID NOs: 2 and 4, and odd sequence identificationnumbers in SEQ ID NOs: 6-47, in which one or more amino acids have beenadded, deleted, substituted, and/or inserted;

(16] a DNA comprising the nucleotide sequence of SEQ ID NO: 5;

(17] a vector in which a DNA according to any one of [13] to [16] isinserted;

[18] a transformant which retains, in an expressible state, the DNAaccording to any one of (13] to [16] or the vector according to [17];

[19] the transformant according to [18], which is a GPI synthasegene-deficient fungus;

[20] the transformant according to [18], which is a GPI synthasegene-deficient yeast;

[21] a method for producing a protein encoded by a DNA according to anyone of [13] to [16], which comprises the steps of culturing thetransformant according to any one of [18] to [20], and recovering theexpressed protein from the transformant or the culture supernatant;

[22] a method of screening for a compound having antimalarial activity,which comprises the steps of:

(1) contacting a test sample with a GPI synthase gene-deficient fungusexpressing the DNA according to any one of [13] to [16],

(2) assaying the growth of that fungus, and,

(3) selecting a test compound that inhibits the growth of that fungus;

[23] a method of screening for a compound having antimalarial activity,which comprises the steps of:

(1) contacting a test sample with a GPI synthase gene-deficient fungusexpressing the DNA according to any one of [13] to [16],

(2) determining the amount of a GPI-anchored protein transported to thefungal cell walls, and,

(3) selecting a test sample that decreases the amount of theGPI-anchored protein transported to the cell wall, as determined in step(2);

[24] a method of screening for a compound having antimalarial activity,which comprises the steps of:

(1) introducing the DNA according to any one of [13] to [16] into a GPIsynthase gene-deficient fungus and expressing the protein encoded by theDNA,

(2) preparing the protein expressed in step (1),

(3) contacting the prepared protein with a test sample and a labeledcompound that has the activity of binding to the protein,

(4) detecting the labeled compound that binds to the protein, and,

(5) selecting a test sample that decreases the amount of labeledcompound that binds to the protein; and,

[25] a method of screening for a compound having antimalarial activity,which comprises the steps of:

(1) introducing into a GWT1-deficient fungus, (i) a DNA encoding aprotein that has the activity of complementing the phenotype of aGWT1-deficient yeast, wherein the DNA is a degenerate mutant of a DNAencoding a malaria parasite GWT1 protein that has a lower AT contentthan the original DNA, or (ii) a vector into which the degenerate mutantof DNA has been inserted, and expressing the protein encoded by thedegenerate mutant DNA,

(2) preparing the protein expressed in step (1),

(3) contacting the prepared protein with a test sample,

(4) detecting GlcN-(acyl)PI, and

(5) selecting a test compound that decreases the level of GlcN- (acyl)PI.

The DNA encoding the GWT1 protein of Plasmodium falciparum (PfGWT1) wasisolated for the first time in the present invention. The nucleotidesequence of the DNA encoding the PfGWT1 protein is shown in SEQ ID NO:1, and the amino acid sequence of the PfGWT1 protein is set forth in SEQID NO: 2. In addition, the nucleotide sequence of the DNA encoding theGWT1 protein of Plasmodium vivax (PvGWT1) is shown in SEQ ID NO: 3, andthe amino acid sequence of the PvGWT1 protein is set forth in SEQ ID NO:4.

The GWT1 protein is involved in the biosynthesis ofglycosylphosphatidylinositol (GPI), which is essential for the growthand infectivity of malaria parasites. Thus, compounds that inhibit theactivity of the malaria parasite GWT1 protein can be used asantimalarial drugs. Such antimalarial drugs can be screened using thismalaria parasite GWT1 protein.

The present invention provides DNAs encoding the malaria parasite GWT1protein. Such DNAs include DNA encoding a protein comprising the aminoacid sequence of SEQ ID NO: 2 or 4, and DNA comprising the nucleotidesequence of SEQ ID NO: 1 or 3.

The present invention also provides DNAs encoding proteins that arefunctionally equivalent to the protein comprising the amino acidsequence of SEQ ID NO: 2 or 4. Herein, the expression “functionallyequivalent” refers to having biological properties equivalent to thoseof the protein of interest, comprising the amino acid sequence of SEQ IDNO: 2 or 4 (the PfGWT1 or PvGWT1 proteins). The biological properties ofthe PfGWT1 and PvGWT1 proteins include GlcN-PI acyltransferase activity.The GlcN-PI acyltransferase activity can be measured by the methodreported by Costello and Orlean (J. Biol. Chem. (1992) 267:8599-8603),or Franzot and Doering (Biochem. J. (1999) 340:25-32).

DNAs encoding proteins functionally equivalent to the protein comprisingthe amino acid sequence of SEQ ID NO: 2 or 4 include: DNAs thathybridize under stringent conditions to the DNA comprising thenucleotide sequence of SEQ ID NO: 1 or 3, and DNA encoding a proteinwhich comprises the amino acid sequence of SEQ ID NO: 2 or 4, in whichone or more amino acids have been added, deleted, substituted, and/orinserted.

The DNAs of the present invention can be isolated by methods well knownto those skilled in the art. Examples of such methods include the use ofhybridization (Southern E. M., J. Mol. Biol. 98: 503-517, 1975) and thepolymerase chain reaction (PCR) (Saiki R. K. et al., Science230:1350-1354, 1985; Saiki R. K. et al., Science239: 487-491, 1988). Morespecifically, it would be routine experimentation for those skilled inthe art to isolate, from malaria parasites, a DNA highly homologous toDNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3, using theDNA of SEQ ID NO: 1 or 3 or portions thereof as a probe, or by using asa primer a DNA which specifically hybridizes to the DNA comprising thenucleotide sequence of SEQ ID NO: 1 or 3. Furthermore, DNAs that can beisolated by hybridization or PCR techniques, and that hybridize with theDNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3, are alsocomprised in the DNAs of the present invention. Such DNAs include DNAencoding a malaria parasite homologue of the protein comprising theamino acid sequence of SEQ ID NO: 2 or 4. The malaria parasite homologueincludes those of Plasmodium falciparum, Plasmodium vivax, Plasmodiummalariae, and Plasmodium ovale, which comprise the amino acid sequenceof SEQ ID NO: 2 or 4.

Preferably, a DNA described above is isolated using hybridizationreactions under stringent hybridization conditions. As used herein, theexpression “stringent hybridization conditions” refers to, for example,hybridization in 4×SSC at 65° C. followed by washing in 0.1×SSC at 65°C. for one hour. Alternative stringent conditions are hybridization in4×SSC containing 50% formamide at 42° C. Further alternative stringentconditions are hybridization in PerfectHyb™ (TOYOBO) solution at 65° C.for 2.5 hours, followed by washing: (1) in 2×SSC containing 0.05% SDS at25° C. for five minutes; (2) in 2x SSC containing 0.05% SDS at 25° C.for 15 minutes; and (3) in 0.1×SSC containing 0.1% SDS at 50° C. for 20minutes. The DNA thus isolated is expected to encode a polypeptide witha high homology at the amino acid level to the amino acid sequence ofSEQ ID NO: 2 or 4. Herein, “high homology” means a sequence identity ofat least 70% or more, preferably 80% or more, more preferably 90% ormore, and most preferably 95% or more, in the whole amino acid sequence.

The degree of identity at the amino acid sequence level or nucleotidesequence level can be determined using the BLAST algorithm of Karlin andAltschul (Karlin S. and Altschul S. F, Proc. Natl. Acad. Sci. USA. 87:2264-2268, 1990; Karlin S. and Altschul S. F, Proc. Natl. Acad. Sci.USA. 90: 5873-5877, 1993). BLAST algorithm-basedprograms, called BLASTNand BLASTX, have been developed (Altschul S. F. et al., J. Mol. Biol.215: 403, 1990). When a nucleotide sequence is analyzed using BLASTN,the parameters are set, for example, at score=100 and word length=12. Onthe other hand, when an amino acid sequence is analyzed using BLASTX,the parameters are set, for example, at score=50 and word length=3. Whenthe BLAST and Gapped BLAST programs are used, the default parameters foreach program are used. Specific procedures for such analysis are known(please see the web site of the National Institute of BiotechnologyInformation http://www.ncbi.nlm.nih.gov).

DNAs of the present invention comprise genomic DNAs, cDNAs, andchemically synthesized DNAs. A Genomic DNA or cDNA can be preparedaccording to conventional methods known to those skilled in the art. Forexample, a genomic DNA can be prepared as follows: (i) extracting agenomic DNA from malaria parasites; (ii) constructing a genomic library(using, for example, a plasmid, phage, cosmid, BAC, or PAC, as avector); (iii) spreading the library; and then (iv) conducting colonyhybridization or plaque hybridization using probes prepared based on aDNA which encodes the malaria parasite GWT1 protein of the presentinvention (e.g., SEQ ID NO: 1 or 3). Alternatively, genomic DNA can beprepared by PCR, using primers specific to a DNA which encodes themalaria parasite GWT1 protein of the present invention. (e.g., SEQ IDNO: 1 or 3). On the other hand, cDNA can be prepared, for example, asfollows: (i) synthesizing cDNA based on mRNA extracted from malariaparasites; (ii) constructing a cDNA library by inserting the synthesizedcDNA into vectors such as XZAP; (iii) spreading the cDNA library; and(iv) conducting colony hybridization or plaque hybridization asdescribed above. Alternatively, the cDNA can also be prepared using PCR.

The present invention also provides DNAs encoding proteins structurallysimilar to the protein comprising the amino acid sequence of SEQ ID NO:2 or 4. Such DNAs include those which comprise nucleotide sequencesencoding proteins comprising amino acid sequences in which one or moreamino acid residues are substituted, deleted, inserted, and/or added.There is no limitation on the number and site of the amino acid mutationin proteins mentioned above, so long as the mutated protein retainsfunctions of the original protein such as those described in Mark, D. F.et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666;l Zoller, M. J.& Smith, M., Nucleic Acids Research (1982) 10, 6487-6500; Wang, A. etal., Science 224, 1431-1433; Dalbadie-McFarland, G. et al., Proc. Natl.Acad. Sci. USA (1982) 79, 6409-6413. The percentage of mutated aminoacids is typically 10% or less, preferably 5% or less, and morepreferably 1% or less of the total amino acid residues. In addition, thenumber of mutated amino acids is usually 30 amino acids or less,preferably 15 amino acids or less, more preferably five amino acids orless, still more preferably three amino acids or less, even morepreferably two amino acids or less.

It is preferable that the mutant amino acid residue be one that retainsthe properties of the side-chain after its mutation (a process known asconservative amino acid substitution). Examples of amino acid side chainproperties are hydrophobicity (A, I, L, M, F, P, W, Y, V) andhydrophilicity (R, D, N, C, E, Q, G, H, K, S, T). Side chains include:aliphatic side-chains (G, A, V, L, I, P); side chains containing anhydroxyl group (S, T, Y); side chains containing a sulfur atom (C, M);side chains containing a carboxylic acid and an amide (D, N, E, Q);basic side-chains (R, K, H); and aromatic side-chains (H, F, Y, W).

A fusion protein comprising the malaria parasite GWT1 protein is anexample of a protein to which one or more amino acids residues have beenadded. Fusion proteins can be made by techniques well known to a personskilled in the art. For example, and without limitation to thisparticular technique, the DNA encoding the malaria parasite GWT1 proteinof the present invention can be combined with DNA encoding anotherpeptide or protein such that their reading frames match. A protein ofthe present invention can form a fusion protein with a number of knownpeptides. Such peptides include FLAG (Hopp, T. P. et al., Biotechnology(1988) 6, 1204-1210), 6×His, 10×His, Influenza agglutinin (HA), humanc-myc fragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag,E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, andProtein C fragment. Examples of proteins that may be fused to a proteinof the present invention include glutathione-S-transferase (GST), HA,immunoglobulin constant region, β-galactosidase, and maltose-bindingprotein (MBP).

In addition to using the above-mentioned hybridization and PCRtechniques, those skilled in the art could prepare the above-describedDNA by methods including, for example, site-directed mutagenesis tointroduce mutations in that DNA (Kramer W. and Fritz H-J., MethodsEnzymol. 154: 350, 1987). A protein's amino acid sequence may also bemutated in nature due to mutation of the nucleotide sequence whichencodes the protein. In addition, degenerate mutant DNAs, in whichnucleotide mutations do not result in amino acid mutations in theproteins (degeneracy mutants), are also comprised in the presentinvention. Furthermore, the present invention also comprises proteinsencoded by the above-described DNAs of this invention.

The present invention provides vectors containing the DNAs of thepresent invention, transformants retaining the DNAs or vectors of thepresent invention, and methods for producing proteins of the presentinvention which utilize these transformants.

A vector of the present invention is not limited so long as the DNAinserted into the vector is stably retained. For example, pBluescript®vector (Stratagene) is preferable as a cloning vector when using E. colias a host. An expression vector is particularly useful when using avector to produce a protein of the present invention. The expressionvector is not specifically limited, so long as it expresses proteins invitro, in E. coli, in cultured cells, and in vivo. Preferable examplesof expression vectors include the pBEST vector (Promega Corporation) forin vitro expression, the pET vector (Novagen) for expression in E. coli,the pME18S-FL3 vector (GenBank Accession No. AB009864) for expression incultured cells, and the pME18S vector (Mol. Cell Biol. 8: 466-472, 1988)for in vivo expression. The insertion of a DNA of the present inventioninto a vector can be carried out by conventional methods, for example,by a ligase reaction using restriction enzyme sites (Current Protocolsin Molecular Biology, ed. by Ausubel et al.,. John Wiley & Sons, Inc.1987, Section 11.4-11.11).

The host cell into which the vector of the present invention isintroduced is not specifically limited, and various host cells can beused according to the objectives of this invention. For example, cellsthat can be used to express the proteins include, but are not limitedto, bacterial cells (e.g., Streptococcus, Staphylococcus, E. coli,Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast,Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animalcells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanomacell), and plant cells. The transfection of a vector to a host cell canbe carried out by conventional methods such as calcium phosphateprecipitation, electroporation (Current protocols in Molecular Biology,ed. by Ausubel et al., John Wiley & Sons, Inc. 1987, Section 9.1-9.9),the Lipofectamine method (GIBCO-BRL), and microinj ection.

By incorporating an appropriate secretion signal into the protein ofinterest, the protein expressed in host cells can be secreted into thelumen of the endoplasmic reticulum, into cavities around the cells, orinto the extracellular environment. These signals may be endogenous orexogenous to the protein of interest.

When a protein of the present invention is secreted into the culturemedium, it is collected from that medium. If a protein of the presentinvention is produced intracellularly, the cells are lysed and then theprotein is collected.

A protein of the present invention can be collected and purified from arecombinant cell culture using methods known in the art, including, butnot limited to, ammonium sulfate or ethanol precipitation, acidextraction, anionic or cationic exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, and lectinchromatography.

Compounds including DNAs of the present invention are isolatedcompounds. Herein, the term “isolated” refers to being separated fromthe original environment (for example, the natural environment if it isnaturally-occurring). A compound in a sample where the compound ofinterest is substantially abundant, and/or in a sample where thecompound of interest has been partially or substantially purified, is an“isolated” compound. The term “substantially purified”, as used herein,refers to a state where the compound has been separated from theoriginal environment, and from which at least 60%, preferably 75%, andmost preferably 90% of other coexisting natural components have beenremoved.

The present invention provides an antimalarial drug that inhibits theactivity of the GWT1 gene product of malaria parasites. A preferredcompound inhibiting the activity of the GWT1 gene product of malariaparasites is the compound described in WO 02/04626, and includes thecompounds (1) to (5):

compound (1): 1-(4-butyl benzyl)isoquinoline

compound (2): 4-[4-(1-isoquinolyl methyl)phenyl] -3-butyne-1-ol

compound (3): 5-butyl-2-(1-isoquinolyl methyl)phenol

compound (4): 2-(4-bromo-2-fluorobenzyl)-3-methoxypyridine

compound (5): N-[2-(4-butyl benzyl)-3-pyridyl]-N-methylamine

A Compound that inhibits the activity of the malaria parasite GWT1 geneproduct, or a salt thereof, or a hydrate thereof, can be administered asit is to mammals (preferably humans). It can also be formulated by aconventional method into a tablet, powder, fine granule, granule, coatedtablet, capsule, syrup, troche, inhalant, suppository, injection,ointment, eye ointment, eye drop, nasal drop, ear drop, cataplasm,lotion, and such, and then administered.

For formulation of a pharmaceutical, auxiliary agents ordinarily used inpharmaceutical formulations (for example, fillers, binders, lubricants,coloring agents, flavoring agents, and as necessary, stabilizers,emulsifiers, absorbefacient, surfactants, pH regulators, antiseptics,and antioxidants) can be used. A pharmaceutical formulation can beprepared using an ordinary method combining components that aregenerally used as ingredients for pharmaceutical preparations.

For example, oral formulations can be produced by combining a compoundof the present invention or a pharmaceutically acceptable salt thereofwith a filler, and as necessary, a binder, disintegrator, lubricant,coloring agent, flavoring agent, and such, and then formulating themixture into a powder, fine granule, granule, tablet, coated tablet,capsule, and such by usual methods.

Examples of these components include: animal fat and vegetable oils suchas soybean oil, beef tallow, and synthetic glyceride, hydrocarbons suchas liquid paraffin, squalene, and solid paraffin; ester oils such asoctyldodecyl myristate and isopropyl myristate; higher alcohols such ascetostearyl alcohol and behenyl alcohol; silicone resin; silicone oil;surfactants such as polyoxyethylene fatty acid ester, sorbitan fattyacid ester, glycerol fatty acid ester, polyoxyethylene sorbitan fattyacid ester, polyoxyethylene hardened castor oil, and polyoxyethylenepolyoxypropylene block copolymer; water-soluble macromolecules such ashydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer,polyethylene glycol, polyvinyl pyrrolidone, and methyl cellulose; loweralcohols such as ethanol and isopropanol; polyhydric alcohols such asglycerol, propylene glycol, dipropylene glycol, and sorbitol; sugarssuch as glucose and sucrose; inorganic powder such as silicic acidanhydride, magnesium aluminum silicate, and aluminum silicate; andpurified water. Examples of fillers include lactose, corn starch,refined white sugar, glucose, mannitol, sorbitol, crystalline cellulose,and silicon dioxide. Binders are polyvinyl alcohol, polyvinyl ether,methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin,shellac, hydroxypropylmethyl cellulose, hydroxypropyl cellulose,polyvinyl pyrrolidone, polypropyleneglycol polyoxyethylene blockpolymer, meglumine, and such. Examples of disintegrators include starch,agar, powdered gelatin, crystalline cellulose, calcium carbonate, sodiumhydrogencarbonate, calcium citrate, dextrin, pectin, and calciumcarboxymethylcellulose. Lubricants are magnesium stearate, talc,polyethyleneglycol, silica, hardened vegetable oil, and such. Examplesof coloring agents are those accepted for addition to pharmaceuticals.Flavoring agents are cocoa powder, 1-menthol, aromatic dispersant, mintoil, borneol, cinnamon powder, and such. The use of sugar coating andother appropriate coating as necessary is of course permissible forthese tablets and granules.

Furthermore, liquid formulations such as syrups and injections can beprepared using conventional methods. In such methods, pH regulators,solubilizers, isotonizing agents, and such, and as necessarysolubilizing adjuvants, stabilizers, and so on, are added to thecompounds of this invention or pharmaceutically acceptable saltsthereof.

Methods for producing external formulations is not restricted and can bea conventional method. That is, base materials used for formulation canbe selected from various materials ordinarily used for medicaments,quasi-drugs, cosmetics, and such. Specifically, the base materials to beused are, for example, animal fat and vegetable oils, mineral oils,ester oils, waxes, higher alcohols, fatty acids, silicone oils,surfactants, phospholipids, alcohols, polyhydric alcohols, water solublemacromolecules, clay minerals, and purified water. As necessary, pHregulators, antioxidants, chelating agents, antiseptic and antifungalagents, coloring matters, fragrances, and such may also be added.However the base materials of the external formulations of the presentinvention are not limited thereto. Furthermore, as necessary, componentssuch as those that have a differentiation-inducing effect, blood flowaccelerants, fungicides, antiphlogistic agents, cell activators,vitamins, amino acids, humectants, and keratolytic agents can becombined. The above-mentioned base materials are added in an amount thatleads to the concentration usually used for external formulations.

The term “salt” as described in the present invention preferablyincludes, for example, a salt with an inorganic or organic acid, a saltwith an inorganic or inorganic base, or a salt with an acidic or basicamino acid. In particular, a pharmaceutically acceptable salt ispreferable. Acids and bases form salts at an appropriate ratio of 0.1 to5 molecules of acid or base to one molecule of the compound.

Preferable examples of a salt with an inorganic acid are a salt withhydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, andphosphoric acid. Preferably, a salt with an organic acid includes a saltwith acetic acid, succinic acid, fumaric acid, maleic acid, tartaricacid, citric acid, lactic acid, stearic acid, benzoic acid,methanesulfonic acid, and p-toluenesulfonic acid.

Preferable examples of a salt with an inorganic base are: an alkalimetal salt such as a sodium salt and a potassium salt; an alkaline earthmetal salt such as a calcium salt and a magnesium salt; an aluminumsalt, and an ammonium salt. Preferably, a salt with an organic baseincludes a salt with diethylamine, diethanolamine, meglumine, andN,N′-dibenzylethylenediamine.

Preferable examples of a salt with an acidic amino acid are a salt withaspartic acid and glutamic acid, and preferably, a salt with a basicamino acid includes a salt with arginine, lysine, and ornithine.

The compounds of the present invention or salts thereof, or hydratesthereof can be administered orally or parenterally by a conventionalmethod without limitation as to their form. They can be formulated intodosage forms such as tablets, powders, fine granules, capsules, syrups,troches, inhalants, suppositories, injections, ointments, eye ointments,eye drops, nasal drops, ear drops, cataplasms, and lotions. The dose ofthe pharmaceutical compositions of this invention can be selectedappropriately depending on the degree of the symptoms, the patient'sage, sex and weight, the dosage form, the type of salt, the specifictype of disease, and such.

Compounds of the present invention are administered to a patient in atherapeutically effective dose. Herein, “therapeutically effective dose”refers to the amount of pharmaceutical agent that yields the desiredpharmacological result and is effective in the recovery or relief fromthe symptoms of the patient to be treated. The dose differs markedlydepending on the type of disease, the degree of symptoms, the patient'sweight, age, sex, sensitivity to the agent. However, the normal adultdosage for one day is about 0.03 mg to 1000 mg, preferably 0.1 mg to 500mg, more preferably 0.1 mg to 100 mg, when administered from once toseveral times a day, or from once to several times over several days.The dose for injections is normally, about 1 to 3000 μg/kg, and ispreferably about 3 to 1000 μg/kg.

In addition, the present invention relates to a method of screening forantimalarial drugs using the malaria parasite GWT1 gene product. Such ascreening method includes, but is not limited to:

[1] A binding assay which screens for compounds that compete with alabeled compound to bind with the malaria parasite GWT1 gene product;

[2] A GlcN-PI acyltransferase assay system to screen for compounds thatinhibit the GlcN-PI acyltransferase activity of the malaria parasiteGWT1 gene product; and [3] A GPI-anchored protein detection system inwhich the malaria parasite GWT1 gene product is expressed in cells,preferably fungal cells, and then the GPI-anchored proteins on the cellsurface are detected. The present invention is not limited to thesemethods, and comprises any method of screening for antimalarial drugsusing the malaria parasite GWT1 gene product. The methods [1] to [3]listed above are described below in detail.

[1] A binding assay to screen for compounds that compete with a labeledcompound to bind with the malaria parasite GWT1 gene product

The two methods according to the present invention are disclosed below,namely (1) a method for preparing the malaria parasite GWT1 gene product(hereinafter referred to as the malaria parasite GWT1 protein) and (2) amethod for a binding experiment involving a labeled compound(hereinafter referred to as a binding assay).

(1) Method for Preparing the Malaria Parasite GWT1 Protein

The malaria parasite GWT1 protein is prepared from a cell membranefraction, preferably from fungal cells, more preferably,from cells of S.cerevisiae into which the DNA encoding the malaria parasite GWT1 proteinof SEQ ID NO: 2 has been introduced. It is preferable to introduce sucha DNA into GWT1 gene-deficient cells. In the binding assay, the preparedmembrane fraction may be used without any further treatment, or can befurther purified before use. The procedure using S. cerevisiae isdescribed below in detail.

(a) Introduction of the Malaria Parasite GWT1 Gene

The malaria parasite GWT1 gene used in the present invention can be anaturally-occurring gene, or preferably, it can be synthesized based onthe amino acid sequence of SEQ ID NO: 2 or 4. The malaria parasite GWT1gene is very rich in adenine and thymine. Thus, it was predictable thatthe gene will be difficult to manipulate with ordinary generecombination techniques, and that gene expression in yeast, cells, andsuch will be inefficient. Therefore, it is preferable to design anucleotide sequence in which codons corresponding to each of thecorresponding amino acids have been replaced with those that are thoughtto express efficiently in yeast, cells, and such, and conduct DNAsynthesis based on this designed sequence to create an artificialmalaria parasite GWT1 gene, which is then used in the experimentsdescribed below.

An expression plasmid for the malaria parasite GWT1 is prepared byinserting the malaria parasite GWT1 gene into an S. cerevisiaeexpression vector, for example, an expression vector prepared byinserting a suitable promoter and terminator, such as the pKT10-derivedGAPDH promotor and GAPDH terminator, into the expression vector YEp352'smulti-cloning site (Tanaka et al., Mol. Cell Biol., 10:4303-4313, 1990).S. cerevisiae (e.g., G2-10 strain) is cultured in an appropriate medium(e.g., YPD medium (Yeast extract-Polypeptone-Dextrose medium)) whileshaking at an appropriate temperature (e.g., 30° C.), and the cells areharvested during the late logarithmic growth phase. After washing, theGWT1-expression plasmid is introduced into S. cerevisiae cells using,for example, the lithium-acetate method. This method is described in theUser Manual of YEAST MAKER™ Yeast Transformation System (BD BiosciencesClontech). A malaria parasite GWT1-overexpressing strain and a straincarrying a negative control vector can be obtained by culturing thetransformed cells on SD (ura-) medium at 30° C. for two days.

Expression vectors and gene transfer methods for fungal species otherthan S. cerevisiae have been reported as follows: expression vectorssuch as pcL for Schizosaccharomyces pombe (S. pombe) and their transfermethods are described by Igarashi et al. (Nature 353:80-83, 1991);expression vectors such as pRM10 for C. albicans and their transfermethods are described by Pla J. et al. (Yeast, 12: 1677-1702, 1996);expression vectors such as pAN7-1 for A. fumigatus and their transfermethods are described by Punt P. J. et al. (GENE, 56: 117-124, 1987);and expression vectors such as pPM8 for C. neoformans and their transfermethods are described by Monden P. et al. (FEMS Microbiol. Lett., 187:41-45, 2000).

(b) Method for Preparing Membrane Fractions

S. cerevisiae cells in which the malaria parasite GWT1 gene has beenintroduced are cultured in an appropriate medium (e.g., SD (ura-) liquidmedium) while being shaken at an appropriate temperature (e.g., 30° C.).The fungal cells are harvested during the mid-logarithmic growth phase,washed, and then suspended in an appropriate amount (e.g., three timesthe volume of fungal cells) of homogenization buffer (e.g., 50 mMTris-HCl, pH 7.5, 10 mM EDTA, Complete™ (Roche)). An appropriate amountof glass beads (e.g., four times the volume of fungal cells) is added tothe suspension. The mixture is vortexed and then allowed to stand onice. This operation is repeated several times to crush fungal cells.

One milliliter of the homogenization buffer is added to the resultinglysate. The mixture is centrifuged, for example at 2,500 rpm for fiveminutes, to precipitate the glass beads and uncrushed fungal cells. Thesupernatant is transferred to another tube. The tube is centrifuged, forexample at 13,500 rpm for ten minutes, to precipitate a membranefraction (total membrane fraction) comprising organelles. Theprecipitate is suspended in 1 ml of binding buffer (e.g., 0.1 MPhosphate buffer, pH 7.0, 0.05% Tween 20, Complete™ (Roche)), and thencentrifuged, for example, at 2,500 rpm for one minute to removeunsuspended material. The supernatant is then centrifuged, for exampleat 15,000 rpm for five minutes. The precipitate isresuspendedin 150to650 μl of binding buffer to prepare a membrane fraction.

Membrane fractions can be prepared from fungal species other than S.cerevisiae using the method of Yoko-o et al. for S. pombe (Eur. J.Biochem. 257:630-637, 1998); the method of Sentandreu M et al. for C.albicans (J. Bacteriol., 180: 282-289, 1998); the method of MouynaI etal. for A. fumigatus (J. Biol. Chem., 275:14882-14889, 2000); and themethod of Thompson J R et al. for C. neoformans (J. Bacteriol., 181:444-453, 1999).

Alternatively, the malaria parasite GWT1 protein can be prepared byexpressing an E. coli, insect and mammalian cell or the like innon-fungal cells.

When mammalian cells are used, the malaria parasite GWT1 gene is ligatedwith an over-expression vector containing, for example, the CMVpromotor, and then introduced into the mammalian cells. Membranefractions can then be prepared according to the method of Petaja-Repo etal. (J. Biol. Chem., 276:4416-23, 2001).

Insect cells expressing the malaria parasite GWT1 gene (e.g., Sf9 cells)can be prepared using, for example, a baculovirus expression kit such asthe BAC-TO-BAC® Baculovirus Expression system (Invitrogen). Membranefractions can then be prepared according to the method of Okamoto et al.(J. Biol. Chem., 276:742-751, 2001).

The malaria parasite GWT1 protein can be prepared from E. coil by, forexample, ligating the malaria parasite GWT1 gene into an E. coliexpression vector such as the pGEX vector (Pfizer Inc.), and introducingthe construct into E. coli such as BL21.

(2) Binding Assay Methods

(a) Synthesis of Labeled Compound

The labeled compound is prepared from a compound that has been confirmedto bind to GWT1 proteins. Any compound which can bind to GWT1 proteinscan be used. The labeled compound is preferably prepared from thecompound described in WO 02/04626, more preferably from compoundsaccording to (1) to (5) described above.

Any labeling method can be used. Preferably, the compound is labeledwith a radioisotope, more preferably with ³H. The radiolabeled compoundcan be prepared by typical production methods using a radioactivecompound as a starting material. Alternatively, ³H labeling can beachieved using an ³H exchange reaction.

(b) Confirmation of Specific Binding

The labeled compound is added to the prepared membrane fraction and themixture is allowed to stand on ice for an appropriate time, for example,one to two hours, while the binding reaction between the labeledcompound and the membrane fraction takes place. The membrane fraction isprecipitated by centrifuging the mixture, for example at 15,000 rpm forthree minutes. The precipitate is resuspended in binding buffer, and thesuspension is centrifuged. This is repeated appropriately (twice) toremove any unbound labeled compound. The precipitate is again suspendedin binding buffer. The resulting suspension is transferred into ascintillation vial, and a scintillator is added. Radioactivity ismeasured using a liquid scintillation counter.

The specific binding of the labeled compound to the GWT1 protein can beconfirmed by assessing whether binding of the labeled compound isinhibited by adding a large excess of unlabeled compound (ten times ormore), and whether the compound binds negligibly to membrane fractionsprepared from fungal cells which do not express the GWT1 protein.

(c) Binding Inhibition of a Labeled Compound by a Test Sample

A test sample and the labeled compound are added to the preparedmembrane fraction, and the mixture is allowed to stand on ice for anappropriate period of time, for example, one to two hours, while thebinding reaction to the membrane fraction takes place. Test compoundsused in the present invention's screening method include: a simplenaturally-occurring compound, an organic compound, an inorganiccompound, a protein, or a peptide, as well as a compound library, anexpression product of a genetic library, a cell extract, a cell culturesupernatant, a product from fermentative bacteria, an extract of amarine organism, a plant extract, and the like.

The mixture is centrifuged, for example at 15,000 rpm for three minutesto precipitate the membrane fraction. The precipitate is resuspended inbinding buffer and the suspension is centrifuged. This is repeatedappropriately (twice) to remove any unbound labeled compound. Theprecipitate is suspended in the binding buffer. The suspension istransferred into a scintillation vial, and scintillator is addedthereto. The radioactivity is measured using a liquid scintillationcounter.

When the binding of the labeled compound to the membrane fraction isinhibited in the presence of a test sample, the test sample is judged tohave the activity of binding to the malaria parasite GWT1 protein.

[2] The GlcN-PI acyltransferase assay system for screening compoundsthat inhibit the GlcN-PI acyltransferase activity of the malariaparasite GWT1 protein

The transfer of an acyl group to GPI can be detected by the methodreported by Costello L. C and Orlean P., J. Biol. Chem. (1992)267:8599-8603; or Franzot S. P and Doering T. L., Biochem. J. (1999)340:25-32. A specific example of the method is described below. Thefollowing experimental conditions are preferably optimized for eachmalaria parasite GWT1 protein to be used.

The malaria parasite GWT1 protein is prepared according to the proceduredescribed in Section 1. Amembrane fraction comprising the malariaparasite GWT1 protein is added to a buffer which comprises anappropriate metal ion (Mg²⁺, Mn²⁺), ATP, Coenzyme A, and preferably aninhibitor that prevents the consumption of UDP-GlcNAc in otherreactions, for example, nikkomycin Z as an inhibitor of chitinsynthesis, or tunicamycin as an inhibitor of asparagine-linkedglycosylation. A test sample is then added to the mixture and theresulting mixture is incubated at an appropriate temperature for anappropriate period of time (for example, at 24° C. for 15 min).

A GlcN-(acyl)PI precursor (for example UDP-GlcNAc, Acyl-Coenzyme A, andpreferably UDP-[¹⁴C]GlcNAc) which has been appropriately labeled, andpreferably radiolabeled, is added to the mixture. The resulting mixtureis incubated for an appropriate period of time (for example, at 24° C.for one hour). A mixture of chloroform and methanol (1:2) is added, theresulting mixture is stirred to halt the reaction, and the lipids areextracted. The extracted reaction product is dissolved in an appropriatesolvent, preferably butanol. Then, GlcN-(acyl)PI produced in thereaction is separated by a method such as HPLC or thin layerchromatography (TLC), preferably TLC. When TLC is used, the developercan be selected appropriately from, for example, CHCl₃/CH₃OH/H₂O(65:25:4), CHCl₃/CH₃OH/1M NH₄OH (10:10:3), and CHCl₃/pyridine/HCOOH(35:30:7). A preferred developer is CHCl₃/CH3OH/H₂O (65:25:4). Theseparated GlcN- (acyl) PI is quantified using a method appropriate forthe label used. When labeled with an radioisotope, the separated GlcN-(acyl) PI can be quantified based on its radioactivity.

When the amount of GlcN-(acyl)PI produced is reduced in the presence ofa test sample, the test sample is judged to have the activity ofinhibiting acyl group transfer by the malaria parasite GWT1 protein.

[3] A GPI-anchored protein detection system which comprises expressingthe malaria parasite GWT1 protein in cells and detecting theGPI-anchored protein on the cell surface

The ability of a test sample to inhibit the activity of the malariaparasite GWT1 protein can be determined using a GPI-anchored proteindetection system that comprises expressing the GWT1 protein in cells,preferably fungal cells, and then detecting the GPI-anchored protein onthe cell surface. The fungi of the present invention are those belongingto Zygomycota, Ascomycota, Basidiomycota, and Deuteromycete, andpreferably pathogenic fungi, Mucor, Saccharomyces, Candida,Cryptococcus, Trichosporon, Malassezia, Aspergillus, Trichophyton,Microsporum, Sporothrix, Blastmyces, Coccidioides, Paracoccidioides,Penicillinium, and Fusarium, more preferably C. albicans, C. glabrata,C. neoformans, and A. fumigatus, and even more preferably, yeast. Suchyeasts include S. cerevisiae and S. pombe. The method for introducinginto the above-described fungal cells an expression vector containinginserted DNA encoding the malaria parasite GWT1 protein is known tothose skilled in the art.

When the malaria parasite GWT1 protein is expressed in fungal cells, theamount of GPI-anchored protein transported to the fungal cell wall canbe determined by the following methods: (1) by using a reporter enzyme;(2) by using an antibody that reacts with the surface glycoprotein offungal cell walls; (3) by using the protein's ability to adhere toanimal cells; or (4) by observing fungal cells under a light microscopeor electron microscope.

The methods of (1) to (4) have been disclosed in WO 02/04626, which isdescribed specifically in Examples of this invention. The methods (1) to(4), and preferably a combination of these methods (1) to (4), candetermine whether a test sample inhibits the transport of theGPI-anchored protein onto the cell wall, or the expression of theGPI-anchored protein on the fungal cell surface.

Hereinafter, the methods of (1) to (4) will be described.

(1) A Method Using a Reporter Enzyme

The process that transports GPI-anchored proteins to the cell wall canbe quantified using a tracer experiment such as one where a GPI-anchoredprotein is labeled with a radioactive isotope, the fungal cell wallfraction is obtained, and immunoprecipitated using an antibody againstthe GPI-anchored protein. Alternatively, quantification can be morereadily performed as follows: the C-terminal sequence, which isconsidered to function as a transport signal and is commonly observedamong GPI-anchored proteins, can be expressed as a fusion protein withan easily measurable enzyme (reporter enzyme), the fungal cell wallfraction can be obtained,; and a reporter system that measures theenzyme activity of each fraction can be used (Van Berkel MAA et al.,FEBS Letters, 349: 135-138, 1994). Hereinafter, a method which uses areporter enzyme will be described, but in the present invention suchmethods are not to be construed as being limited thereto.

First, the reporter gene is constructed and introduced into fungi. Thereporter gene is constructed by linking a promoter sequence thatfunctions in fungi with DNAs that respectively encode a signal sequence,a reporter enzyme, and a GPI-anchored protein C-terminal sequence insuch a way that the reading frames match. Examples of the promotersequence are GAL10 and ENO1. Examples of the signal sequence includeα-factor, invertase, and lysozyme. Examples of reporter enzymes areβ-lactamase, lysozyme, alkaline phosphatase, and β-galactosidase. GreenFluorescence Protein (GFP), which has no enzyme activity but can beeasily detected, can also be used. GPI-anchored protein C-terminalsequences include the α-agglutinin C-terminal sequence, the CWP2C-terminal sequence, and so on. Furthermore, it is preferable to insertan appropriate selection marker, such as LEU2 and URA3, into the vectorcomprising the constructed reporter gene.

The constructed reporter gene is inserted into fungi using anappropriate method, such as the lithium acetate method (Gietz D et al.,Nucl. Acids Res. 20: 1425, 1992). The fungi are then cultured, asnecessary, using a method that suits the selection marker (e.g. usingLeu medium for LEU2 and Ura⁻ medium for URA3), and then fungi into whichthe DNA has been introduced are selected.

The effect of a test sample on the transport of GPI-anchored proteins tothe cell wall is examined by the following method:

The reporter gene-introduced fungi are cultured under appropriateconditions, for example at 30° C. for 48 hours, in the presence of atest sample. After culturing, the culture supernatant is centrifuged,and the reporter enzyme activity of the culture supernatant fraction ismeasured. The resulting cell fraction is washed, the cell wallcomponents are separated using an appropriate method, such as degradingthe cell wall glucan with glucanase, and then the reporter enzymeactivity of the cell wall fraction and cytoplasmic fraction is measured.The assay can be simply carried out by using centrifugation to determinethe amount of reporter enzyme in the cell fraction, then without washingthe cells, using proportional calculations to determine the amount ofreporter enzyme derived from the culture supernatant fraction thatremains in the cell fraction, and subtracting this from the amount ofreporter enzyme of the cell fraction.

If the test sample exhibits the activity of increasing reporter enzymeactivity within the culture supernatant fraction (activity per cell), orthe activity of decreasing the reporter enzyme activity in the cell wallfraction (activity per cell), the test sample is judged to haveinfluenced the transport process of GPI-anchored proteins to the cellwall.

(2) A Method Using an Antibody That Reacts With the Surface Glycoproteinof Fungal Cell Walls

A test sample s ability to influence the expression of a GPI-anchoredprotein at the fungal surface layer can be determined by quantificationusing an antibody that reacts with that GPI-anchored protein in thefungal cell wall.

Antibodies can be obtained by predicting the antigenic determinant usingthe amino acid sequence of, for example, a GPI-anchored protein such asα-agglutinin, Cwp2p, or Als1p (Chen M H et al., J. Biol. Chem.,270:26168-26177, 1995; Van Der Vaat J M et al., J. Bacteriol.,177:3104-3110, 1995; Hoyer L L et al., Mol. Microbiol., 15:39-54, 1995),and then synthesizing the peptide of that region, binding it to anantigenic substance such as a carrier protein, and then immunizing arabbit or such to obtain polyclonal antibodies, or a mouse or such toobtain a monoclonal antibody. A rabbit polyclonal antibody against theAls1p peptide is preferable.

In an alternative method, a monoclonal antibody against a GPI-anchoredprotein may be obtained by immunizing mice and such with fungi,preferably fungi which overexpress a GPI-anchored protein such asα-agglutinin, Cwp2p, and Als1p, (in some cases by immunizing furtherwith a partially purified GPI-anchored protein), and then using ELISA,Western blot analysis, and so on to select resultant clones based on theantibody that they produce.

The following method can be used to determine the influence of a testsample on the process that transports a GPI-anchored protein to the cellwall, and on the amount of protein derived from that GPI-anchoredprotein in the cell wall.

Fungi are cultured in the presence of a test sample under appropriateconditions such as 30° C. for 48 hours. The cultured fungi are collectedby centrifugation and the cells are disrupted, preferably using glassbeads. The washed, disrupted cells are preferably subjected tocentrifugal extraction with SDS, and then the precipitate is washed.After extraction, the disrupted cells are treated with an enzyme thatdegrades glucan, preferably glucanase, and the centrifuged supernatantthereof is the GPI-anchored protein sample.

The anti-Alslp peptide antibody is coated onto a 96-well plate byovernight incubation at 4° C. The plate is washed with a washingsolution, preferably PBS comprising 0.05% Tween 20 (PBST), and blockingis carried out using a reagent that blocks the non-specific adsorptionsites of the 96-well plate, preferably a protein such as BSA or gelatin,more preferably BlockAce (Dainippon Pharmaceutical Co.,Ltd.). The plateis again washed with a washing solution, preferably PBST, and anappropriately diluted GPI-anchored protein sample is added. The reactionis then carried out for an appropriate time such as two hours at roomtemperature. After washing with a washing solution, preferably withPBST, an antibody against the enzyme-labeled C. albicans, preferablyHRP-labeled anti-Candida antibody, is reacted for an appropriate timesuch as two hours at room temperature. The labeling method may be enzymelabeling or radioactive isotope labeling. After washing with a washingsolution, preferably PBST, the amount of Alslp in the GPI-anchoredprotein sample is calculated by a method appropriate to the type oflabel, i.e. for an enzyme label, by adding a substrate solution andthen, upon stopping the reaction, measuring absorbance at 490 nm.

(3) A method Using the Ability to Adhere to Animal Cells

The test sample's influence on the expression of a GPI-anchored proteinon the fungal surface can be determined by measuring the activity ofthat GPI-anchored protein in the fungal cell wall, and preferably bymeasuring the ability of fungi to adhere to animal cells and the like.In addition to the activity of Als1p, Hwp1p and such in adhesion toanimal cells, GPI-anchored protein activity includes that ofα-agglutinin in mating, of Flo1p in yeast aggregation, and so on.Hereinafter, a method using the ability of fungi to adhere to animalcells will be described in detail, but the present invention is not tobe construed as being limited thereto.

A fungus with the ability to adhere to cells is used, and this fungus ispreferably C. albicans. For mammalian cells, cells that adhere to thefungus, preferably intestinal epithelial cells, are used. The mammaliancells are cultured and fixed using an appropriate method, such asethanol fixation. The test sample and the fungi are incubated for anappropriate time such as 48 hours at 30° C., then inoculated andcultured for a set time, for example, one hour at 30° C. The culturesupernatant is then removed, and the cells are washed with a buffer andoverlaid with agar media such as Sabouraud Dextrose Agar Medium (BectonDickinson Company, Ltd.). After culturing at 30° C. overnight, thenumber of colonies is counted, and the adhesion rate is calculated.

If, when compared to fungi not treated with the compound, a test sampleis observed to have the activity of decreasing the number of coloniesformed by cell adhesion, that test sample is judged to have influencedthe process that transports GPI-anchored proteins to the cell wall.

(4) A Method for Observing Fungi Using an Electron Microscope or anOptical Microscope

The influence of a test sample on the expression of the GPI-anchoredprotein in the fungal surface can be determined by observing thestructure of the fungal cell wall using an electron microscope.

In the presence of a test sample, a fungus such as C. albicans iscultured for a certain period of time, for example, 48 hours at 30° C.,and its ultrafine morphological structure is observed using atransmission electron microscope. Herein, observation using atransmission electron microscope can be carried out, for example by themethod according to the Electron Microscope Chart Manual (MedicalPublishing Center). The flocculent fibrous structure of the outermostlayer of a fungal cell has a high electron density and is observable bytransmission electron microscope. This structure is not influenced byother existing antifungal agents and is considered to be a surfaceglycoprotein layer, including GPI-anchored proteins as its constituents.When this structure disappears, leaving only a slight layer with a highelectron density, the test sample is judged to have influenced theprocess that transports GPI-anchored proteins to the cell wall, comparedto untreated cells.

When observation under both a transmission electron microscope and anoptical microscope reveals greatly swollen fungal cells and inhibitedbudding (division), the test sample is judged to have an influence onthe cell wall.

The present invention also provides a method for treating malaria, whichcomprises the step of administering a compound that inhibits theactivity of a GWT1 protein a malaria parasite. Such a compound includesthe compounds described in WO 02/04626 (for example, the compoundsdescribed herein in (1)-(5)).

The nucleotide sequence for the natural PfGWT1 protein is characterizedby an exceedingly high AT content (80.41%), and thus codon usage isbiased. In addition, the gene contains sequence stretches comprising sixor more consecutive A residues at 23 separate positions, and thesesequence stretches may serve as pseudo-poly(A) sites, thus producingtruncated proteins. Because of the features described above, the genewas only expressed poorly in yeast, and very difficult to amplify usingPCR or to replicate in E. coli. It was also difficult to determine thenucleotide sequence. However, the present inventors succeeded inexpressing the PfGWT1 protein with a high efficiency by using adegenerate mutant of the DNA (SEQ ID NO: 5), with a lower AT contentthan the DNA encoding the PfGWT1 protein. The inventors also revealedthat the introduction of the degenerate mutant DNA can rescue thephenotype of GWT1-deficient yeast. This finding suggests that the GPIsynthase of a malaria parasite is interchangeable with that of a fungussuch as yeast.

The AT content of the gene encoding the malaria parasite GPI synthaseis, for example, 79.35% for GPI8 and 77.89% for the GPI13 of P.falciparum. These AT contents are as high as that of PfGWT1. It ispredicted that most P. falciparum genes are hardly expressed in otherspecies, because the average AT content over the translated regions ofthe P. falciparum genome is 76.3%. The present inventors succeeded inexpressing a degenerate mutant of the DNA with a lower AT content thanthat of the DNA encoding the PfGWT1 protein, in yeast. Hence, themalaria parasite GPI synthase can be expressed in a host other thanmalaria parasites by using such a degenerate DNA mutant. Furthermore,GPI-deficient yeast and GWT1-deficient yeast are known to exhibitsimilar phenotypes, including the characteristic of lethality and such.Thus, the phenotype of the GPI synthase gene-deficient fungus can berescued by using the degenerate mutant DNA described above.

The phenotype of the GPI synthase gene-deficient fungus into which thedegenerate mutant DNA described above has been introduced depends on theactivity of the malaria parasite GPI synthase . Accordingly, compoundsthat inhibit the activity of the malaria parasite GPI synthase can beselected by screening using the phenotype of the GPI synthasegene-deficient fungus as an index. Thus, antimalarial drugs targetingthe GPI biosynthesis pathway can be selected without actually using themalaria parasites themselves.

The present invention provides a degenerate mutant DNA encoding aprotein that has the activity of rescuing the phenotype of a GPIsynthase gene-deficient fungus, and which has an AT content lower thanthat of the original DNA encoding the protein involved in thebiosynthesis of GPI. Such a DNA can be used in the screening method ofthe present invention.

As used herein, the term “AT content” refers to the content of adenineand thymine in the entire nucleotide sequence of the coding region ofthe GPI synthase gene. The AT content in the degenerate mutant DNA ofthe present invention preferably ranges from 50% to 70%, more preferablyfrom 53% to 65%, and still more preferably from 55% to 62%.

The phenotype of the GPI synthase gene-deficient fungus includestemperature sensitivity (preferably, sensitivity to high temperatures)and lethality.

The proteins of the present invention involved in the biosynthesis ofGPI in malaria parasites include GWT1, GPI1, GPI8, GPI3/PIG-A,GPI10/PIG-B, YJR013W/PIG-M, GPI13/PIG-O, GAA1/GAA-1, DPM1, GPI2, GPI15,YDR437W, GPI12, MCD4, GPI11, GPI7, GPI17, GPI16, CDC91, DPM2, DPM3, andSL15. Of the proteins indicated above, GPI1 and GPI8 have been found tobe present in malaria parasites, and GPI3/PIG-A, GPI10/PIG-B,YJR013W/PIG-M, GPI13/PIG-O, GAAl/GAA-1, and DPM1 have been suggested tobe present in malaria parasites (Delorenzi et al., Infect. Immun. 70:4510-4522, 2002). The nucleotide sequences of GWT1, GPI1, GPI8,GPI3/PIG-A, GPI10/PIG-B, YJR013W/PIG-M, GPI13/PIG-O, GAA1/GAA-1, andDPM1 of P. falciparum are shown in SEQ ID NO: 1 and the even sequenceidentification numbers in SEQ ID NOs: 6-21, respectively. Eachcorresponding amino acid sequence is shown in SEQ ID NO: 2 and the oddsequence identification numbers in SEQ ID NOs: 6-21. In addition, thenucleotide sequence of P. vivax GWT1 is shown in SEQ ID NO: 3, and thecorresponding amino acid sequence is shown in SEQ ID NO: 4. Using amethod known to those skilled in the art, for example, a method usinghybridization or PCR, GWT1, GPI1, GPI8, GPI3/PIG-A, GPI10/PIG-B,YJRO13W/PIG-M, GPI13/PIG-O, GAA1/GAA-1, or DPM1 of other malariaparasites can be cloned using DNA comprising any one of the nucleotidesequences shown in SEQ ID NO: 1 and 3, and the even-numbered SEQ ID NOs:6-21.

Furthermore, GPI synthase genes other than GWT1, GPI1, GPI8, GPI3/PIG-A,GPI10/PIG-B, YJR013W/PIG-M, GPI13/PIG-O, GAA1/GAA-1, and DPM1 of malariaparasites can be cloned by using yeast or human GPI synthase genes. Thenucleotide sequences of GPI2, GPI15, YDR437W, GPI12, MCD4, GPI11, GPI7,GPI17, GPI16, and CDC91 of yeast (S. cerevisiae) are shown in the evensequence identification numbers in SEQ ID NOs: 22-41 respectively; andeach corresponding amino acid sequence is shown in the odd sequenceidentification numbers in SEQ ID NOs: 22-41. In addition, the nucleotidesequences of human DPM2, DPM3, and SL15 are shown in the even sequenceidentification numbers in SEQ ID NOs: 42-47 respectively; and eachcorresponding amino acid sequence is shown in the odd sequenceidentification numbers in SEQ ID NOs: 42-47.

The production of a degenerate mutant DNA encoding a protein involved inthe biosynthesis of the GPI of malaria parasites, and with a lower ATcontent than that of the original DNA, consists of two steps: design,and synthesis. In the design step, the amino acid sequence of a proteinof interest is first reverse-translated and then possible codons foreach amino acid residue are listed. Reverse translation can be achievedby using commercially available gene analysis software (for example,DNASIS-Pro; Hitachi Software Engineering Co., Ltd). Of the codonslisted, those meeting the purpose (for example, codons whose AT contentis lower and codons frequently used in the host to be used for geneexpression) are selected for each amino acid. The degenerate mutant DNAcan be designed by rearranging the amino acid sequence of the protein ofinterest using these selected codons.

The DNA thus designed can be synthesized by a method known to thoseskilled in the art. The degenerate mutant DNA of the present inventioncan be synthesized based on the designed nucleotide sequence by, forexample, using a commercially available DNA synthesizer.

The present invention also provides vectors in which the above-describeddegenerate mutant DNA has been inserted, and transformants (preferablyGPI synthase gene-deficient fungi) that retain the DNA or the vector inan expressible state. The vector and the host can be those describedabove.

As used herein, the expression “deficient in the GPI synthase gene”means that the functional product of the gene is not expressed, or thatthe expression level is decreased. The GPI synthase gene-deficientfungus of the present invention can be prepared by disrupting the GPIgene. The disruption can be achieved by inserting DNA unrelated to thegene, for example a selection marker, based on homologous recombinationtechnology, and the like. More specifically, such a mutant fungus can beprepared by introducing into yeast a selection marker cassette whichcomprises the his5 gene or the kanamycin resistance gene of S. pombe(Longtine et al., Yeast, 14: 953-961, 1998) amplified with primers, eachof which comprises a nucleotide sequence homologous to a portion of thegene (ranging from 50 to 70 nucleotides).

The GPI synthase gene-deficient fungus of the present inventionincludes, for example, the GWT1 temperature-sensitive mutant straingwt1-20, GPI7 disruptant strain, GPI8 mutant strain gpi8-1, and GPI10temperature-sensitive mutant strain per13-1.

A GPI synthase gene-deficient fungus which has been transformed with thedegenerate mutant DNA of the present invention can be prepared byintroducing into a fungus a vector into which the degenerate mutant DNAhas been inserted. pRS316, YEp351, or such can be used as the vector forS. cerevisiae, and pcL, pALSK, or such can be used as the vector for S.pombe.

The present invention also provides a method of screening forantimalarial drugs, which comprises using GPI synthase gene-deficientfungi described above.

In such a method, the first step comprises contacting a test sample witha GPI synthase gene-deficient fungus that has been transformed withdegenerate mutant DNA with a lower AT content than the DNA encoding aprotein involved in the biosynthesis of GPI of malaria parasites. The“contact” can be achieved by adding a test sample to the culture of theabove-mentioned fungus. When the test sample is a protein, a vectorcomprising DNA encoding the protein can be introduced into theabove-mentioned fungus.

In the method of the present invention, the next step comprisesmeasuring the degree of growth of the above-mentioned fungus. Morespecifically, the fungus is inoculated under typical culture conditions,specifically, the fungus is inoculated onto a liquid culture medium suchas Yeast extract-polypeptone-dextrose medium (YPD medium) or onto anagar plate, and then incubated at 25 to 37° C. for 4 to 72 hours. ThusGPI synthase gene-deficient fungus transformed with the degeneratemutant DNA of the present invention can be assessed for growth. Thedegree of growth can also be determined using the turbidity of theculture liquid, the number of colonies, or the size or color of thespots formed on the agar plate as an index. In the method of the presentinvention, the next step comprises selecting compounds that inhibit thegrowth of the above-mentioned fungus.

In an alternative method, the first step comprises contacting a testsample with a GPI synthase gene-deficient fungus in which theabove-described degenerate mutant DNA has been introduced. The next stepcomprises determining the amount of GPI-anchored protein transportedonto the yeast cell wall. The detection method includes: (1) methodsusing a reporter enzyme; (2) methods using an antibody that reacts witha surface glycoprotein on the fungal cell wall; (3) methods using theability to adhere to animal cells; and (4) methods using a lightmicroscope or an electron microscope to observe the fungi. In the methodof the present invention, the next step comprises selecting a samplethat decreases the amount of GPI-anchored protein transported to thecell wall.

The present invention provides a method of screening for antimalarialdrugs using a protein involved in the biosynthesis of GPI, which isprepared using a degenerate mutant DNA of the present invention. Suchmethods include, for example, a binding assay system where screening iscarried out to select compounds that bind to a protein involved in GPIbiosynthesis in competition with a labeled compound bound to theprotein. Specifically, a degenerate mutant DNA of the present inventionis introduced into the GPI. synthase gene-deficient fungus, the proteinencoded by the DNA is expressed in the fungus, and the expressed proteinis prepared. The prepared protein is then contacted with a test sampleand with a labeled compound that can bind to the protein. In the nextstep, the labeled compound bound to the protein is detected, and testsamples that decrease the amount of labeled compound bound to theprotein are selected.

The present invention also provides an assay system for GlcN-PIacyltransferase. Such a system comprises using a GWT1 protein which isprepared using a DNA encoding a protein that has the activity ofcomplementing the phenotype of GWT1-deficient yeast, which the DNA is adegenerate mutant of a DNA encoding a malaria parasite GWT1 protein thathas a lower AT content than the original DNA. Specifically, thedegenerate mutant DNA is introduced into GWT1-deficient fungus, theprotein encoded by the degenerate mutant DNA is expressed in the fungus,and the expressed protein is prepared. This protein is then contactedwith a test sample, GlcN-(acyl)PI is detected, and a test sample thatdecreases the amount of GlcN-(acyl) PI is selected.

Any patents, patent applications, and publications cited herein areincorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts photographs showing the results of tetrad analysis. Thegwt1-disrupted strain became viable after the introduction of theopfGWT1-overexpressing plasmid. The four spores derived from a singlediploid cell were spotted vertically.

If one copy of the GWT1 gene was disrupted, only half of the sporesgrew. Thus, the ratio of [colony-forming spots]: [spots exhibiting nogrowth] is 2:2 in such cases. In the columns marked with an arrow, thelethal phenotype of the gwt1 disruptant was complemented by theintroduced opfGWT1, and hence all four spots grew, each forming acolony.

FIG. 2 depicts a diagram showing the inhibitory activity of a compoundwith respect to the growth of yeast expressing the opfGWT1 gene. Eitherthe yeast GWT1 gene or opfGWT1 gene was expressed in GWT1 gene-disruptedyeast.

A compound having the activity of inhibiting the GWT1-dependent growthof yeast also showed inhibitory activity with respect to theopfGWT1-dependent growth of yeast in which opfGWT1 was expressed.

FIG. 3 depicts a diagram showing antimalarial activity. Human red bloodcells were infected with P. falciparum. A GWT1-inhibiting compound wasadded to these red blood cells, and inhibition of malaria parasiteinfection was determined.

All five compounds exhibiting antifungal activity also inhibited themalaria parasite infection of red blood cells.

BEST MODE FOR CARRYING OUT THE INVENTION

Herein below, the present invention will be specifically described usingExamples, but it is not to be construed as being limited thereto.

EXAMPLE 1 P. falciparum GWT1 (PfGWT1)

(1) The nucleotide sequence of P. falciparum GWT1 (PfGWT1) (SEQ IDNO: 1) has been disclosed in the database of the P. falciparum genome(PlasmoDB database, http://plasmodb.org/). The PfGWT1 gene was cloned byPCR using genomic DNA purified from P. falciparum (the 3D7 strain) as atemplate. The 5′ half and 3′ half of the PfGWT1 gene were preparedseparately, and the two halves were assembled at an XbaI (TCTAGA)restriction enzyme site. Thus, the full-length PfGWT1 gene was prepared.In addition, restriction enzymes sites outside the coding region wereincluded, thus allowing insertion into an expression vector.

(2) The 5′ half of the PfGWT1 gene was amplified by PCR using P.falciparum genomic DNA as a template and the primers pf152F (SEQ ID NO:48) and pf136R (SEQ ID NO: 49). The 3′ half was amplified by the sameprocedure described above, using the primers pf137F (SEQ ID NO: 50) andpf151R (SEQ ID NO: 51). The DNA fragments amplified were subcloned intothe pT7-Blue vector (Novagen), and the nucleotide sequences of theinserts were sequenced to confirm homology to SEQ ID NO: 1. Clonescontaining the 5′ half of the PfGWT1 gene were named PF15-5 clones.Clones containing the 3′ half were named PF20-9 clones.

(3) Using PCR, cleavage sites for restriction enzymes were added outsidethe coding region to enable the PfGWT1 gene to be inserted into anexpression vector. An EcoRI cleavage site was added to the 5′ half byPCR using PF15-5 as a template and the primers pf154FE (SEQ ID NO: 52)and pfl57R (SEQ ID NO: 53). The amplified DNA fragment was subclonedinto the pT7-Blue vector (Novagen) to prepare the clone pT7-plasmN2.Likewise, the 3′ half was amplified by PCR using PF20-9 as a templateand the primers pf168BK (SEQ ID NO: 54) and pf155RK (SEQ ID NO: 55). Theamplified DNA fragments were subcloned to prepare pT7-plasmBK5 clones.

(4) The full-length PfGWT1 gene was prepared by the procedure describedbelow. The yeast expression vector YEp352GAPII was digested with therestriction enzymes EcoRI and KpnI. The EcoRI-XbaI fragment (about 1500bp) derived from pT7-plasmN2, and the XbaI-KpnI fragment (about 1100 bp)derived from pT7-plasmBK5, were inserted into the vector at a cleavedsite. The expression vector YEp352GAPII-PfGWT1 containing thefull-length PfGWT1 was then constructed. [pf152F]ATGACAATGTGGGGAAGTCAACGGg (SEQ ID NO: 48) [pf136R]TGTGTGGTTACCGTTCTTTGAATACATAGA (SEQ ID NO: 49) [pf137F]ATAGAAAATGATTTATGGTACAGCTCAAA (SEQ ID NO: 50) [pf151R]AGACCAAATTAATTATGCCTTTACATGTAC (SEQ ID NO: 51) [pf154FE]agaattcaccATGAGCAACATGAATATACTTGCGTATCTT (SEQ ID NO: 52) [pf157R]GAAATTCCAATGTATTCCATATTCACTTAT (SEQ ID NO: 53) [pf168BK]AAGATCTAATACATTAAAACATTTTAGATTAATGAATATGTG (SEQ ID NO: 54) [pf155RK]aggtaccGTACACTCCACTCTATGATGATCATTC (SEQ ID NO: 55)

EXAMPLE 2 A Fully Synthetic PfGWT1 Gene

The adenine and thymine (AT) proportion is exceedingly high (80% orhigher) in P. falciparum DNA, and thus routine biological techniques(PCR, E. coli-based gene engineering, expression systems for recombinantproteins, and so on) are often unavailable (Sato and Horii; Protein,Nucleic acid, and Enzyme Vol. 48, 149-155, 2003). Likewise, the ATcontent of PfGWT1 DNA was 80.41% including many consecutive A or Tstretches. Thus, the gene was predicted to be difficult to replicate andexpress as a protein in yeast. Indeed, when native PfGWT1 ligated with ayeast overexpression vector was introduced into GWT1 disrupted yeast,the PfGWT1 did not rescue the lethal phenotype of the GWT1 disruptant atall. To reduce AT content, codons were replaced with synonymous codonswithout changing the original amino acid sequence.

The codon substitution was carried out based on the nucleotide sequenceof P. falciparum GWT1 (SEQ ID NO: 1) disclosed in the P. falciparumgenome database (PlasmoDB database, http://plasmodb.org/). The resultingnucleotide sequence was named “optimized PfGWT1 (opfGWT1)” (SEQ ID NO:5).

The sequence described above was designed to include additionalsequences outside the coding region; namely an EcoRI cleavage sitesequence (GAATTC, at the5′ end), Kozak's sequence (ACC, at the 5′end),and a KpnI cleavage site sequence (GGTACC, at the 3′ end). The synthesisof the resulting sequence was consigned to Blue Heron Inc. in the U.S.A.These additional restriction enzyme sites were used to ligate the fullysynthetic opfGWT1 into the YEp352GAPII vector to construct anoverexpression plasmid for opfGWT1. The construct was introduced intodiploid cells (WDG2) in which only a single copy of the GWT1 gene hadbeen disrupted. The resulting transformants were cultured on platescontaining a sporulation medium to form spores for tetrad analysis.

The AT content of the newly designed codon-modified opfGWT1 was reducedto 61.55%. The results of tetrad analysis are shown in FIG. 1. Thegwt1-disrupted strain became viable after introduction of the opfGWT1overexpression plasmid. The findings described above indicate that thePfGWT1 gene can be expressed in yeast cells when its AT content isreduced by codon modification.

EXAMPLE 3 An Assay for Antimalarial Activity Using opfGWT1-ExpressingYeast

A screening system for compounds having antimalarial activity wasconstructed using opfGWT1-expressing yeast.

An expression cassette was constructed by inserting the S. cerevisiaeGWT1 terminator, and the S. cerevisiae GAPDH promoter and multi-cloningsite into the SacI-KpnI site of the single-copy vector pRS316. S.cerevisiae GWT1 and opfGWT1 were inserted into the multi-cloning site toprepare pGAP-ScGWT1 and pGAP-opfGWT1 plasmids, respectively. Theseplasmids were introduced into the GWT1 disruptant. Serial two-folddilutions of compound (1) were prepared using YPAD to make the highestfinal concentration 50 μg/ml. A 50 μl aliquot of the diluted compoundwas added to each well of a 96-well plate. Overnight cultures of yeastcells comprising each plasmid were diluted 1000-fold and then a 50 μlaliquot of the dilution was added to each well. The plates wereincubated at 30° C. for two days, and then culture turbidity wasdetermined at 660 nm (FIG. 2 and Table 1). TABLE 1 0 6.25 12.5 25 50pGAP-ScGWT1 0.7560 0.7370 0.6670 0.1140 0.0420 pGAP-opfGWT1 0.71500.6990 0.6910 0.3630 0.0530

Although the GWT1 disruptant was nonviable, the strain became viableafter introduction of each plasmid (as shown at 0 μg/ml of compoundconcentration). The growth of ScGWT1-expressing yeast was inhibited bycompound (1), a GWT1-specific inhibitor. The use of the compound at 25μg/ml resulted in about 85% inhibition of growth. When the compound wasused at 50 μg/ml, the yeast was completely nonviable. The growth ofopfGWT1-expressing yeast was also inhibited by compound (1). The use ofthe compound at 25 μg/ml resulted in about 50% inhibition of growth.When the compound was used at 50 μg/ml, the yeast was completelynonviable. Since growth of opfGWT1-expressing yeast depends on theactivity of the introduced opfGWT1, growth inhibition can be attributedto the inhibition of the opfGWT1 function by compound (1). Thesefindings suggest that compounds with P. falciparum GWT1-specificinhibitory activity GWT1 can be identified by screening compounds usingthis assay system.

EXAMPLE 4 Antimalarial Activity

Representative compounds (1) to (5), that inhibit yeast GWT1, wereassayed for antimalarial activity using a red blood cell culture system.

compound (1): 1-(4-butyl benzyl)isoquinoline

compound (2): 4-4- (1-isoquinolyl methyl)phenyl]-3-butyne-1-ol

compound (3): 5-butyl-2-(1-isoquinolyl methyl)phenol

compound (4): 2-(4-bromo-2-fluorobenzyl)-3-methoxypyridine

compound (5): N-[2-(4-butyl benzyl)-3-pyridyl]-N-methylamine

Specifically, a test compound was dissolved in 100% DMSO, diluted with amedium, and an 80 μl aliquot of the dilution was added to each well of a96-well culture plate. P. falciparum FCR3 strain was pre-cultured inRPMI1640 medium containing 10% human serum at 37° C., and then 20 μl ofthe cultured cells (containing 10% red blood cells) was added to eachwell. At this time, 0.47% of red blood cells were infected. Afterculturing under 5% O₂, 5% CO₂, and 90% N₂ at 37° C. for 48 hours, themalaria parasites were stained using Giemsa staining. The number ofprotozoan-infected red blood cells was determined in order to estimateinfection rate (FIG. 3). As a result, compound (3) was revealed to havestrong antimalarial activity. The other four compounds also showedantimalarial activity. Compound (4) exhibited the lowest activity.Therefore, compounds inhibiting yeast GWT1 include compounds which havethe activity of inhibiting P. falciparum GWT₁, suggesting thatantimalarial drugs can be synthesized based on such compounds.

INDUSTRIAL APPLICABILITY

The present invention succeeded in producing fungi that express malariaparasite GWT1. Using such fungi, antimalarial drugs targeting thepathway of GPI biosynthesis can be screened without using malariaparasites.

To date, no attempt has been made to express a malaria parasite gene infungal cells and screen substances which inhibit the function of thatgene. The methods of the present invention remove the need to actuallyusing malaria parasites themselves, and thus this method proves thepossibility of entirely new screening methods for drug discovery usingcomparative genomics in the post-genome era.

1. A DNA according to any one of (a) to (d), which encodes a protein ofa malaria parasite having GlcN-PI acyltransferase activity: (a) a DNAencoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or4, (b) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3,(c) a DNA hybridizing to DNA comprising the nucleotide sequence of SEQID NO: 1 or 3 under stringent conditions, and (d) a DNA encoding aprotein which comprises the amino acid sequence of SEQ ID NO: 2 or 4, inwhich one or more amino acids have been added, deleted, substituted,and/or inserted.
 2. A protein encoded by the DNA according to claim 1.3. A vector into which the DNA according to claim 1 is inserted.
 4. Atransformant which retains, in an expressible state, the DNA accordingto claim 1 or a vector comprising the DNA.
 5. An antimalarial drug whichcomprises as an active ingredient a compound that inhibits the activityof the protein according to claim
 2. 6. The antimalarial drug accordingto claim 5, wherein the compound that inhibits the activity of theprotein is at least one selected from the group consisting of thefollowing compounds (1) to (5):


7. A method of screening for a compound having antimalarial activity,which comprises the steps of: (1) contacting the protein according toclaim 2 with a test sample and a labeled compound that has the activityof binding to the protein, (2) detecting the labeled compound that bindsto the protein, and, (3) selecting a test sample that decreases theamount of labeled compound that binds to the protein.
 8. The methodaccording to claim 7, wherein the labeled compound that has the activityof binding to the protein is produced by labeling at least one compoundselected from the group consisting of the following compounds (1) to(5):


9. A method of screening for a compound having antimalarial activity,which comprises the steps of: (1) contacting a test sample with theprotein according to claim 2, (2) detecting GlcN-(acyl)PI, and, (3)selecting a test compound that decreases the level of GlcN-(acyl)PI. 10.A method of screening for a compound having antimalarial activity, whichcomprises the steps of: (1) contacting a test sample with a celloverexpressing the protein according to claim 2, (2) determining theamount of GPI-anchored protein transported to the cell wall in the cell,and, (3) selecting a test sample that decreases the amount of theGPI-anchored protein transported to the cell wall, as determined in step(2).
 11. A method for treating malaria, which comprises administering acompound that inhibits the activity of the protein according to claim 2.12. The method according to claim 11, wherein the compound comprises theantimalarial drug of claim
 5. 13. A DNA encoding a protein that has theactivity of complementing the phenotype of a GPI synthase gene-deficientyeast, which is a degenerate mutant of a DNA encoding a protein involvedin GPI biosynthesis in malaria parasites, and that has a lower ATcontent than the original DNA.
 14. A DNA encoding a protein that has theactivity of rescuing the phenotype of a GPI synthase gene-deficientyeast, which is a degenerate mutant of a DNA encoding a protein involvedin the biosynthesis of GPI in malaria parasites, and which has an ATcontent of 70% or less.
 15. The DNA according to claim 13 or 14, whichis selected from the group consisting of: (a) a DNA encoding a proteinthat comprises any one of the amino acid sequences of SEQ ID NOs: 2 and4, and odd sequence identification numbers in SEQ ID NOs: 6-47, (b) aDNA comprising any one of the nucleotide sequences of SEQ ID NOs: 1 and3, and even sequence identification numbers in SEQ ID NOs: 6-47, (c) aDNA hybridizing under stringent conditions to the DNA that comprises anyone of the nucleotide sequences of SEQ ID NOs: 1 and 3, and evensequence identification numbers in SEQ ID NOs: 6-47, and (d) a DNAencoding a protein which comprises any one of the amino acid sequencesof SEQ ID NOs: 2 and 4, and odd sequence identification numbers in SEQID NOs: 6-47, in which one or more amino acids have been added, deleted,substituted, and/or inserted.
 16. A DNA comprising the nucleotidesequence of SEQ ID NO:
 5. 17. A vector in which the DNA according to anyone of claims 13, 14 or 16 is inserted.
 18. A transfornant whichretains, in an expressible state, the DNA according to any one of claims13, 14 or 16, or a vector comprising the DNA.
 19. The transformantaccording to claim 18, which is a GPI synthase gene-deficient fungus.20. The transformant according to claim 18, which is a GPI synthasegene-deficient yeast.
 21. A method for producing a protein encoded bythe which comprises the steps of culturing the transformant according toclaim 18, and recovering the expressed protein from the transformant orthe culture supernatant.
 22. A method of screening for a compound havingantimalarial activity, which comprises the steps of: (1) contacting atest sample with a GPI synthase gene-deficient fungus that expresses theDNA according to any one of claims 13, 14 or 16, (2) assaying the growthof that fungus, and, (3) selecting a test compound that inhibits thegrowth of that fungus.
 23. A method of screening for a compound havingantimalarial activity, which comprises the steps of: (1) contacting atest sample with a GPI synthase gene-deficient fungus expressing the DNAaccording to any one of claims 13, 14 or 16, (2) determining the amountof a GPI-anchored protein transported to the fungal cell wall, and, (3)selecting a test sample that decreases the amount of the GPI-anchoredprotein transported to the cell wall, as determined in step (2).
 24. Amethod of screening for a compound having antimalarial activity, whichcomprises the steps of: (1) introducing the DNA according to any one ofclaims 13, 14 or 16 into a GPI synthase gene-deficient fungus andexpressing the protein encoded by the DNA, (2) preparing the proteinexpressed in step (1), (3) contacting the prepared protein with a testsample and a labeled compound that has the activity of binding to theprotein, (4) detecting the labeled compound that binds to the protein,and, (5) selecting a test sample that decreases the amount of labeledcompound that binds to the protein.
 25. A method of screening for acompound having antimalarial activity, which comprises the steps of: (1)introducing into a GWT1-deficient fungus, (i) a DNA encoding a proteinthat has the activity of complementing the phenotype of a GWT1-deficientyeast, wherein the DNA is a degenerate mutant of a DNA encoding amalaria parasite GWT1 protein that has a lower AT content than theoriginal DNA, or (ii) a vector into which the degenerate mutant of DNAhas been inserted, and expressing the protein encoded by the degeneratemutant DNA, (2) preparing the protein expressed in step (1), (3)contacting the prepared protein with a test sample, (4) detectingGlcN-(acyl)PI, and (5) selecting a test compound that decreases thelevel of GlcN-(acyl)PI.